Interest in angiogenesis is evidenced by the vast amount of literature available on the subject, some of which is over one hundred years old. See, e.g., Virchau, R., Die Krankhaftern Geshwulste, Hirshwald, Berlin (1863); Thierisch, C., Die Hautmit Altas, Leipzig (1865). "Angiogenesis" is defined as the process by which new blood vessels are formed, with accompanying increased blood circulation. Much of the research in this field over the past ten years has concentrated on identifying and purifying naturally occurring substances which cause angiogenesis. Examples of the literature in this area include Weiss, et al., Br. J. Cancer 40: 493-96 (1979); Fencelau, et al., J. Biol. Chem. 256: 9605-9611 (1981); McAslan, et al., Exp. Cell Res. 119: 181-190 (1979), which show that angiogenic factors are present in tissues of pathological origin, such as tumor cells. Kuma,, et al., Lancet 2: 364-367 (1983); and Brown, et al., Lancet 1: 682-685 (1980), show an angiogenesis factor in synovial fluid of arthritis patients, while Hill, et al., Experentia 39: 583-585 (1983) show one in vitreous of arthritic patients. Banda, et al., Proc. Natl. Acad. Sci. 79: 7773-7777 (1982), teach one in wound fluid. Additional teachings in this field include those of D'Amore, et al., Proc. Natl. Acad. Sci. 78: 3068-3072 (1981); Kissun, et al., Br. J. Ophthalmol. 66: 165-159 (1982); DeCarvellho, et al., Angiology 34: 231-243 (1983); Frederick, et al., Science 224: 289-290 (1980); Burgos, Eur. J. Clin. Invest 13: 289-296 (1983); and Catellot, et al., Proc. Natl. Acad. Sci. 79: 5597-5601 (1982), all of which show the existence of angiogenic factors in normal tissues.
Characteristic of all of the angiogenic materials discussed in the above referenced prior art is that they are involved in normal growth and development. In other words, angiogenesis is a necessary process during the growth and development of an individual organism. The prior art angiogenic factors described supra are involved in these normal processes, but are apparently not implicated in enhanced rates of angiogenesis, which is sometimes necessary or desirable.
One of the first endeavors involving investigation of lipid factors provoking enhanced angiogenesis is to be found in Goldsmith, et al., JAMA 252: 2034-2036 (1984). The factor was found in chloroform-methanol fractionates of feline omentum. This research is presented in U.S. Pat. No. 4,699,788, the disclosure of which is incorporated herein. This extract is a lipid extract.
Additional research based upon the teachings of Goldsmith, et al. found that the class of glycolipids known as gangliosides possesses enhanced angiogenic activity. This may be seen in U.S. Pat. No. 4,710,490, the disclosure of which is incorporated by reference herein.
It will be seen that the newest research on angiogenesis points to lipid containing molecules as provoking enhanced angiogenesis. This was new to the art, as the earlier literature all suggested that protein derived material caused angiogenesis. Thus, Kumar, et al., Lancet 2: 364-367 (1983) teach proteins of from 300 to 10.sup.5 daltons, while Kissun, et al., Br. J. Ophthalmol 66: 165-169 (1982), show protein factors weighing up to 70 kilodaltons. Banda, et al., Proc. Natl. Acad. Sci. 79: 7773-7777 (1982), teach proteins of from 2 to 14 kilodaltons as provoking angiogenesis and Burgos, et al., Eur. J. Clin. Invest 13: 289-296 (1983), show protein complexes of from 100 to 200 kilodaltons. A very recent report, by van Brunt, et al., Biotechnology 6(1): 25-30 (Jan. 1988) describes angiogenesis caused by proteins.
The art, however, contains no mention of omega-3 polyunsaturated fatty acids as angiogenic substances.
The class of lipid molecules known as "omega 338 , ".omega.-3" or "n-3" polyunsaturated fatty acids ("n-3 PUFAs") have been studied in various contexts. Omega-3 is a shorthand notation that designates the positional arrangement of the double bonds closest to the terminal methyl group (FIG. 1).
The omega-3 family is obtained mainly from dietary seafood or can be derived from linolenic acids which occur in leafy vegetables and unhydrogenated soybean oil. The levels of omega-3 in human tissues are low except for those people consuming large quantities of seafood and in particular the 20:5.omega.3 (EPA) and 22:6.omega.3 (DHA). Evidence shows that members of the Greenland Eskimo population, who consume a diet rich in seafood, as compared to their conterparts living in Denmark, have a higher amount of omega-3 present. (Dyerberg, J. 1981, Phil trans. Royal Soc. London B294: 373; Jorgensen K. A. and Dyerberg, J. 1982 Adv. Nutr. Res. 5:57; Lee, 1985 et al, New Eng. J. Med. 312:1217).
Much evidence shows that a diet rich in omega-3 fatty acids has beneficial effects in humans, including a reduction in plasma cholesterol and triglycerides levels, improved fat tolerance, prolonged bleeding time, reduced platelet counts and decreased platelet adhesiveness (Lee et al, supra; Phillpson et al, 1985 New Eng. J. Med. 312: 1210). Of primary interest is the effect of these fatty acids on heart disease. Thus, Kinsella, in Food Technology: 89-97, 146 (February 1986), summarizes the literature on n-3 PUFAs as obtained from fish. This reference explores the various biochemical pathways in which the n-3 PUFAs participate, and suggests some of the reasons why these materials may be involved in alleviating ischemic heart disease. In a similar vein, Saynor, et al., Atheriosclerosis 50: 3-10 (1984) discuss the role of one n-3 PUFA, i.e., eicosapentaenoic acid ("EPA") in reducing triglyceride and very low density lipoprotein (VLDL) serum levels. The paper also shows, however, that bleeding time increased, and the ability of platelets to aggregate decreased when EPA was administered.
Terano, et al, Atheriosclerosis 46: 321-331 (1983) comment on the same effects, but also state that EPA reduces whole blood viscosity.
The patent literature on the n-3 or .omega.-3 PUFAs is scant. Rubin, U.S. Pat. No. 4,526,902, teaches the treatment of thrombo-embolic conditions using a combination of one of EPA (5, 8, 11, 14, 17-eicosapentaenoic acid), and DHA (22:6 .omega.-3 docosahexaenoic acid) and a linoleic acid derivative. Of interest in this reference, the content of which is incorporated herein, is the teaching that mixtures of EPA and DHA/linoleic acid derivatives need not be administered as pharmaceuticals, but that they can also be administered in food form, such as cooking oil or margarine. Debat, U.S. Pat. No. 4,186,211, teach that higher alkanols and their esters can be used to treat prostate conditions. No .omega.-3 PUFAs are taught; rather docosa- and eicosaalkanol derivatives are the active compositions.
It will be seen then, that none of the prior art teaches or suggests that omega-3 polyunsaturated fatty acids may be used to provoke or to enhance angiogenesis.
Hence it is an object of the invention to provide a method for provoking or enhancing angiogenesis in an individual subject by administering to the subject an angiogenically effective amount of an angiogenically active .omega.-3 polyunsaturated fatty acid. Especially preferred are methods of treatment utilizing eicosapentaenoic acid (EPA), and docosahexaenoic acid (DHA). How the objects of this invention are achieved will be seen via review of the disclosure which now follows.